Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that manipulates the host immune system and establishes life-long latent infection. This successful co-existence with the host is mediated by the production of viral proteins that mimic normal immune modulators like cytokines, chemokines, and chemokine receptors. HCMV encodes four proteins with similarity to human chemokine receptors, US27, US28, UL33, and UL78. US28 and UL33 both exhibit ligand-independent constitutive signaling activity, and US28 has also been shown to signal in response to cellular chemokines. In contrast, no ligands or signaling outcomes have been identified for US27, and it remains an orphan receptor. A stable cell line expressing US27 was developed previously in order to screen for possible chemokine ligands for US27. While no ligands were identified, we made the unexpected observation that US27 significantly enhanced the signaling of a human chemokine receptor, CXCR4, in response to its natural ligand, CXCL12/SDF-1?. One of the many important physiological roles of CXCR4 involves directing immune cells to the bone marrow, which is the primary site of HCMV latency. Potentiation of CXCR4 signaling by US27 could represent a strategy by which HCMV targets virus-infected cells to the bone marrow in order to initiate a new round of infection and expand the reservoir of latently infected cells. The specific hypothesis being tested is that US27 forms heteromers with CXCR4 that stimulate increased movement of infected cells toward the chemokine CXCL12/SDF-1?. This hypothesis is supported by strong preliminary data demonstrating that cells expressing both US27 and CXCR4 exhibit greater calcium mobilization and enhanced chemotaxis in response to CXCL12/SDF-1? than controls. In addition, US27 and CXCR4 were found to co-localize to discrete locations in the cell by immunofluorescence staining and confocal microscopy. The specific goals of this proposal are to identify the mechanism for increased CXCR4 signaling in the presence of US27 and to determine whether enhanced CXCR4 responses occur in virus-infected cells. The overall goal of the project is to clarify the role of US27 in viral pathogenesi, and the results are expected to reveal a novel regulatory function for an orphan receptor with possible implications for the maintenance of HCMV latency.